Cancer is a term commonly known as a disease occurring due to indiscreet proliferation of cells and due to damage to the mechanism of regulating cell proliferation. In accordance with data released by World Health Organization (WHO) in February, 2012, the number of deaths due to cancer throughout the world in 2008 was about 7.6 million people accounting for 13% of mortality over the world and, and five cancers such as lung cancer, stomach cancer, liver cancer, colon cancer and breast cancer account for about 50% of the entire death rate of cancer. In addition, it was shown that 12.7 million new cancer patients were diagnosed during 2008, and among them, about 70% occurred in underdeveloped/developing countries such as China, South America, Africa, and the like. The occurrence rate of new cancer patients has steadily increased, such that it is expected to reach 22 million cancer patients in 2030.
In particular, the occurrence rate of cancer patients in Asia has rapidly increased from obesity due to westernized diet, an increase in intake of red meat and alcohol, and the like. It is expected that the occurrence rate of cancer patients per 100,000 people will rapidly increase from 122 people in 2005 to 163 people in 2030 by 45% or more, which is about 7.34 million people when converting the rate into the number of patients.
Cancer is a disease currently ranking No. 1 for the cause of death in Korea and the number of new cancer patients for 1 year in 2009 according to data released by Statistics Korea on Dec. 30, 2011 was 192,561 (male: 99,224, female: 93,337), which had increased by 6.7% as compared to 180,465 in 2008 and by 90.6% as compared to 101,032 in 1999. For people in Korea, surviving to the age of 81, which is an average life span of people in Korea, the probability of suffering from cancers is 36.2%, and cancer has progressed in one of three patients (2 out of 5 male, and 1 out of 3 female). It was found that the total number of people with cancer surviving until the end of 2009 was 808,503, and the number of people overcoming cancer or living with cancer was 8 hundred thousand.
In accordance with Global Cancer Facts & Figures, 2nd Edition published by American Cancer Society in 2011, the cost for prevention, diagnosis, and treatment of cancer all over the world was estimated to be about 895 billion US dollars (985 trillion won in Korean money). It was investigated that cost incurred in relation with cancer in the US in 2010 was about 263.8 billion US dollars (290 trillion won in Korean money), the direct medical cost was 102.0 billion US dollars (112 trillion won in Korean money), the loss due to disease was 20.9 billion US dollars (23 trillion won in Korean money), and the economical loss due to premature death was 140.1 billion US dollars (154 trillion won in Korean money).
In accordance with cancer registry statistics released by the National Cancer Center in December, 2011, in Korea, it was estimated that the cost associated with cancer was: liver cancer (mean cost: 66.22 million won), lung cancer (46.47 million won), gastric cancer (26.85 million won) and colorectal cancer (23.52 million won).
A main reason that a normal cell is changed into a cancer cell is due to the abnormality of a gene, wherein the abnormality is caused by genetic factors inherited from parents; however, there are many cases where the abnormality is developed by acquired factors such as carcinogens, smoking, diet, virus infection, and the like. As a result, personal deviation in view of the reaction to chemotherapy after surgical procedure and the recurrence of cancer is largely shown. That is, since cancer is developed by reflecting genetic and environmental factors, cancer has patient-specific properties, and a degree of sensitivity to a specific drug also differs with each patient.
Hierarchial model, which is the latest theory with respect to cells configuring cancer tissue, asserts that a few tumor stem cells are present in tumor tissue and more differentiated cells without self reproduction ability are produced while maintaining a few tumor stem cells (E Passegue C. H. et al., Proc Natl Acad Sci USA, 30; 100 Suppl 1:11842-11849, 2003). Therefore, since a few tumor stem cells capable of inducing cancer and most of the differentiated cells losing cancer inducing ability are mixed, the tumor stem cell has drug resistance with respect to the conventional anti-cancer agents developed by having tumor cells occupying the majority in the cancer tissue as a target. As long as the tumor stem cell is present, the tumor may recur at any time, which is a key point of the tumor stem cell theory.
A theory of tumor stem cell was established by confirming the presence of the tumor stem cell in hematologic malignancy inducing leukemia for the first time in 1997 (Bonnet D. et al., Nat Med., 3(7):730-737, 1997). That is, it was confirmed that when cells determined as a cancer stem cell in an acute myeloid leukemia are extracted and transplanted into an immunosuppressive rat, human-derived leukemia is developed in a rat even with a small amount of cells. Then, evidence that tumor stem cells are present even in solid tumor cancers in breast cancer, colon cancer, prostate cancer, melanoma were suggested (Singh S. K. et al., Nature, 18; 432(7015):396-401, 2004).
The tumor stem cell has similar properties as a stem cell, for example, in the case of the acute myeloid leukemia, wherein cells having CD34+CD38-phenotype of hematopoietic stem cells have properties of the tumor stem cell (Bonnet D. et al., Nat Med., 3(7):730-737, 1997). In addition, brain tumor stem cells and normal nerve cells commonly express CD133, wherein CD133+ brain cancer cells even with 100 or less of a small amount thereof forms a tumor in the cranial cavity of a rat (Singh S. K. et al., Nature, 18; 432(7015):396-401, 2004; Kondo T. et al, Proc Natl Acad Sci USA, 20; 101(3):781-786, 2004). As another example of properties similar to stem cell, it is known that the brain tumor cell forms a sphere under specific conditions, like a nerve stem cell (Sanai N. et al., N Engl J Med., 353: 811-822, 2005). In addition, when serum is added to the brain tumor stem cell, the brain tumor stem cell may be similarly differentiated to the nerve stem cell (Rao J. S., Nat Rev Cancer, 3:489-501, 2003).
Meanwhile, in the case where expected effectiveness of an anti-cancer agent known to have excellent effects is not shown due to patient-specificity of cancer, trial and error for an appropriate treatment is inevitable, and risk and burdens of patients are increased. Thus, when a trial for reflecting personal characteristics in development and screening of an anti-cancer agent has been actively conducted, and it has been verified through various clinical tests that in the case where a customized targeted treatment having a target as a specific patient group is performed, treatment reaction of the patient is better than that of the existing standard anti-cancer treatment (Alterovitz G. et al., Oral Oncol., 47(10):951-5, 2011; Arnedos M. et al., Mol Oncol., 6(2):204-10, 2012; Black A. and Morris D., Curr Oncol., 19(Suppl 1):S73-85, 2012).
The beginning of the existing systematic drug screening method included injection of a mouse leukemia cell into an abdominal cavity and analysis of treatment effect of the drug at National Cancer Institute (NCI) in 1955 and establishment of a human cancer cell line xenograft transplantation model using immunodeficiency mouse established in the 1970s enables screening of main solid carcinoma (Hausser H. J. et al., Biochem Biophys Res Commun., 333:216-2, 2005). In 1989, NCI converted the basis of the screening strategy from a compound to a disease and then introduced an NCI-60 cell line panel consisting of human cancer cell lines having various histological and genetic properties, wherein in a retrospective analysis with respect to 39 drugs achieving up to clinical phase 2, in the case where over ⅓ of subcutaneous xenograft transplantation models show efficacy, it was reported that there is a significant relationship with a treatment reactivity in an actual patient (De Wever O. et al., J Pathol., 200:429-47, 2003).
However, since the above-described existing drug development system performs a drug screening based on a single cell line cultured in vitro for a long time, there are many cases where an efficacy of a developed drug is different from that in the actual clinical test. It is general that a proliferation assay constructing an anti-cancer agent screening is useful for evaluating an efficacy to an abnormal proliferation ability of a cancer cell; however, such has a limitation in reflecting sensitivity to the drug according to gene information. In particular, since a high throughput anti-cancer agent screening system constructed up to now demands an adherent culture of a cell onto an artifact surface, there is high probability of bringing genotypic and phenotypic changes and it is difficult to represent in an in vivo environment (US Patent Application Publication No. 2012-0077698). In order to overcome the above-described recent limitation, an effort to apply a three-dimensional culture to the high throughput screening system has been conducted; however, there are problems in that many cells are demanded for an analysis, and determination depends on a subjective determination of an inspector. In addition, in the case where the three-dimensional culture is a floating culture, since a focal distance for each area is different from each other, there are limitations in view of time and physical aspect in that each image should be taken from several thousands of wells for an automatic analysis (US Patent Application Publication No. 2009-0221441). Therefore, in order to precisely and rapidly conduct a screening method of anti-cancer materials with respect to various conditions, research into a technology of an automatic analysis method satisfying an environment capable of maximally maintaining properties of cancer cells, a demand on small number of cells, and an objective analysis is required.
Meanwhile, the genetically modified model and the xenograft transplantation model based on cancer cell lines among animal models easily cause a change in the cell lines, such that there are many cases of losing original properties as the cancer cell. In securing a required number of cell lines, the cell lines go through a long-time selection process under in vitro conditions. It was known that the above-described models are homogeneous and undifferentiated as compared to a tissue derived from an actual cancer patient (Hausser H. J. et al., Biochem Biophys Res Commun., 333:216-2, 2005). In addition, since human-derived stromal cells and immune cells configuring a microenvironment around cancer which is important for growth and metastasis of cancer are absent, original biological and molecular properties of a patient carcinoma are not reproduced any more (De Wever O. et al., J Pathol., 200:429-47, 2003).
As a useful method for overcoming the problem, patient-derived tumografts in which surgically removed patient-derived tumor tissue is directly transplanted into an immunodeficiency mouse was suggested (Rubio-Viqueira B. and Hidalgo M., Clin Pharmacol Ther., 85:217-21, 2009; Fichtner I et al., Eur J Cancer., 40:298-307, 2004). Since both of the cancer tissue and the stromal cell of microenvironment around cancer are derived from the same patient, the patient-derived tumorgrafts are evaluated as being that transplantation success, growth of cancer tissue, and reaction to drug are most similarly reproduced. However, about 90% of anti-cancer agents showing a remarkable anti-cancer effect in a preclinical model and entering a clinical test are not capable of reproducing effects in actual patients up to the present.
Therefore, in order to develop a screening method for effectively selecting an optimal anti-cancer agent and an optimal combination of anti-cancer agents with respect to an individual patient, a technology capable of reflecting gene information of a patient, mimicking an environment in a human body, and efficiently analyzing a large amount of samples is needed.
Accordingly, the present inventors made an effort to solve the problems of the related art as described above. As a result, the present inventors confirmed that in the case where a patient-derived cancer cell is subjected to a screening method including three-dimensional culture using a limiting dilution assay, patient-specific anti-cancer agents are capable of being efficiently selected by using an extremely small amount of cells as compared to the existing screening methods, thereby completing the present invention.